Project Summary Despite notable improvements in early detection and therapeutic options, breast cancer has persisted in leading the United States in cancer related mortalities, taking the lives of more than 40,000 Americans each year. Epidermal growth factor receptor (EGFR) inhibitors have been approved as targeted therapies for a number of cancers, which include lung and colorectal cancers, but have seen limited success for breast cancer. In theory, EGFR was identified as a target of interest for breast cancer therapy because its expression levels are negatively correlated with breast cancer patient outcomes and it is highly expressed in many breast cancer cases. However, in practice, it was noted that only a small fraction of patients responded positively to EGFR inhibitor treatment, with many developing resistance to the inhibitors. Fortunately, current research efforts have revealed that this resistance may be attributed to the activity of other receptor tyrosine kinases. Through the support of our own preliminary data, we hypothesize that receptor tyrosine kinase-like orphan receptor 1 (ROR1) is one of the major contributors to EGFR resistance in triple-negative breast cancer patients, likely through activation of YAP pathway. The ligand-bound ROR1 associates with HER3 to trigger previous unidentified signaling cascades, leading to activation of YAP-target transcriptional program. Additionally, the expression of ROR1 and status of HER3 phosphorylation are correlated with unfavorable outcomes for breast cancer patients, and knock out of ROR1 inhibited metastasis of TNBC in preclinical models. Our central hypothesis is that ROR1/HER3-triggered activation of the YAP pathway promotes distal metastasis of TNBC and resistance to EGFR-target therapy. We will address this hypothesis according to the following 3 specific aims. In Specific Aim 1, we will demonstrate the functional role of the ROR1/HER3 signaling axis in metastatic TNBC. In Specific Aim 2, we will define the molecular mechanism that underlies the ROR1- mediated signaling pathway to activate the YAP target transcriptional program. In Specific Aim 3, we will determine the preclinical benefits of targeting ROR1/YAP in combination with EGFR inhibitors. EGFR inhibitor treatment has been successfully implemented into the care plans of patients with a variety of cancer types. However, EGFR inhibitor treatment has only been marginally successful for TNBC patients. The proposed research will investigate the effectiveness of targeting the ROR1/YAP signaling pathway in circumventing resistance to EGFR inhibitors and thus improving response to the widely used inhibitors. The research team will rely on high-throughput sequencing, biochemistry, molecular biology, and xenograft mouse models to comprehensively assess a co-targeting strategy that involves EGFR and ROR1/YAP to help provide the scientific basis for a successful treatment approach for patients with TNBC.